Hales B F, Huang C
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.
Biochem Pharmacol. 1994 Jun 1;47(11):2029-37. doi: 10.1016/0006-2952(94)90078-7.
Manipulation of the glutathione status of an embryo during organogenesis leads to abnormal development, as well as increasing the susceptibility of the embryo to insult by either xenobiotic or endogenous electrophiles. The glutathione S-transferases are a family of drug-metabolizing enzymes that catalyze the conjugation of reactive chemicals with glutathione, playing an important role in protecting cells against attack. The purpose of this study was to investigate the presence and regulation of one glutathione S-transferase, glutathione S-transferase P, a homodimer of the Yp subunit, in the conceptus during organogenesis. Northern blot analysis of the RNA isolated from rat embryos and their yolk sacs on days 10, 11 and 12 of gestation revealed a single Yp transcript. Steady-state concentrations of the Yp mRNA in embryos did not change with either gestational age or culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). In contrast, concentrations of this transcript in yolk sac increased 3-fold from day 10 to 12 of gestation and a further 3-fold with culture (day 12 in vivo compared with in vitro). Transcription of the rat Yp subunit gene in cell lines is induced by treatment with phorbol esters. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 or 100 nM) to embryos in culture had no effect on the steady-state concentrations of the Yp transcript. Thus, the glutathione S-transferase Yp message is subject to tissue- and development-specific regulation in the conceptus during organogenesis. Moreover, culture of the embryos resulted in a further up-regulation of the steady-state concentrations of the Yp transcript in yolk sac. Western blot analysis demonstrated that a single immunoreactive Yp subunit band of 26 kDa was found in both embryos and yolk sacs. Neither age nor culture appeared to affect the concentrations of immunoreactive Yp subunit in the yolk sac. Thus, glutathione S-transferase Yp mRNA is translated in the conceptus during organogenesis. The apparent differences between the relative amounts of the message and immunoreactive protein in yolk sac suggest that this subunit may be subject to post-transcriptional as well as transcriptional regulation in this tissue. Immunohistochemical analysis of embryos cultured for 45 hr (day 12 in vitro) revealed that the Yp reaction product was localized over the hepatic primordia, mesonephric ducts, otocyst, yolk sac and ectoplacental cone.(ABSTRACT TRUNCATED AT 400 WORDS)
在器官发生过程中对胚胎谷胱甘肽状态进行调控会导致发育异常，同时还会增加胚胎对异生物或内源性亲电试剂损伤的易感性。谷胱甘肽S-转移酶是一类药物代谢酶，可催化活性化学物质与谷胱甘肽结合，在保护细胞免受攻击方面发挥重要作用。本研究的目的是调查一种谷胱甘肽S-转移酶——谷胱甘肽S-转移酶P（Yp亚基的同二聚体）在器官发生期间的胚胎中的存在情况及其调控机制。对妊娠第10、11和12天大鼠胚胎及其卵黄囊分离的RNA进行Northern印迹分析，结果显示有一条单一的Yp转录本。胚胎中Yp mRNA的稳态浓度在妊娠年龄、24小时培养（体外第11天）或45小时培养（体外第12天）时均未发生变化。相比之下，卵黄囊中该转录本的浓度在妊娠第10天到第12天增加了3倍，培养后（体内第12天与体外相比）又增加了3倍。细胞系中大鼠Yp亚基基因的转录可通过佛波酯处理诱导。然而，向培养的胚胎中添加12-O-十四酰佛波醇-13-乙酸酯（TPA，50或100 nM）对Yp转录本的稳态浓度没有影响。因此，在器官发生期间的胚胎中，谷胱甘肽S-转移酶Yp信息受到组织和发育特异性调控。此外，胚胎培养导致卵黄囊中Yp转录本的稳态浓度进一步上调。蛋白质印迹分析表明，在胚胎和卵黄囊中均发现一条26 kDa的单一免疫反应性Yp亚基带。年龄和培养似乎都不影响卵黄囊中免疫反应性Yp亚基的浓度。因此，在器官发生期间胚胎中谷胱甘肽S-转移酶Yp mRNA会被翻译。卵黄囊中信息和免疫反应性蛋白质相对量之间的明显差异表明，该亚基在该组织中可能受到转录后以及转录调控。对培养45小时（体外第12天）的胚胎进行免疫组织化学分析显示，Yp反应产物定位于肝原基、中肾管、耳泡、卵黄囊和外胎盘锥。（摘要截短至400字）
